PROJECT SUMMARY Adhesion of lymphocytes to other cells or tissues is a tightly regulated process governed by interactions between lymphocyte protein receptors, including integrins, and their ligands. Integrin binding affinity regulation is critical to cell homing to sites of inflammation and occurs via conformational changes that stem from complex intersubunit. Imbuing similar properties of regulated binding into engineered proteins promises to potentiate the development of novel ?smart? materials that incorporate these molecules, providing coupled environmental responsiveness and adhesion for enhanced delivery of drugs, genes, or imaging agents to appropriate targets. Achieving this potential requires: (1) selection of an appropriate, modular molecular scaffold for regulated target binding and (2) development of a robust protein engineering toolkit for tailoring both sensing and adhesive properties of the scaffold. The PI?s lab has recently developed a novel molecular chassis consisting of the inserted domain (I- domain) of ?L integrin fused with two EF hand subdomains from calmodulin (CaM). The resulting I-domain-CaM chimera demonstrates the ability to bind to ICAM-1, the natural adhesive ligand for I-domain, at levels increased by ~75-fold in the presence of a peptide ligand bound by CaM. In addition, data obtained in the development of this molecular switch suggest additional mechanistic insight into the structural regulation of I-domain. Here, we aim to experimentally explore this structural mechanism and to develop and demonstrate directed evolution tools to engineer the specificity of both key functions (adhesive binding and sensing) of the chimeric switch; the binding specificities of both the I-domain and CaM modules will be retargeted to novel model ligands. This work will set the stage for development of I-domain- CaM fusions engineered for a wide range of practical applications and may provide better understanding of I-domain conformational regulation that will prove relevant to describing and manipulating integrin-based cell adhesion.